Decoy-oligonucleotide-inhibition of cd40-expression

ABSTRACT

The present invention relates to decoy oligonucleotides with the nucleic acid sequence according to SEQ ID NO: 1 to 36 and their use as pharmaceutical agents.

The present invention relates to decoy oligonucleotides with the nucleicacid sequence according to SEQ ID NO: 1 to 36 and their use aspharmaceutical agents.

The transplantation of solid organs generally represents the last resortin the treatment of diseases, in which the organ to be replaced in therecipient's body is severely damaged and/or can no longer adequatelyfulfil its function. This occurs, for example, in the terminal stage ofheart failure, but also in cases of acute or chronic renal or hepaticfailure. The pancreas, lung and small intestine are transplantedroutinely but less frequently than the organs mentioned. A combinedtransplantation of several organs is also possible. In addition to thesolid organs, the cornea of the eyes and haematopoietic stem cells fromthe bone marrow are also transplanted.

A total of 3130 transplantations of solid organs were carried out inGermany in 2000 (Eurotransplant). The major problem in this context isstill the acute rejection of the donor organ by the recipient organism.This rejection reaction (host-versus-graft reaction) is more marked, theless closely the immunological features of the donor agree with those ofthe recipient (lack of histocompatibility). In the presence of adequatehistocompatibility, the rejection reaction can generally be suppressedwith appropriate drugs (immunosuppressants); however, long-termtreatment with these drugs can lead to serious side-effects. Forexample, transplant patients frequently develop tumours and infectionsas a result of their impaired immune defences. The chronic rejection oftransplanted organs is also intensified. This degeneration of thearteries and arterioles supplying the transplanted organ, also referredto as vasculopathy, represents a special, accelerated form ofatherosclerosis (transplant atherosclerosis), which leads successivelyby progressive functional impairment to the failure of the transplantedorgan. If a re-transplantation is not possible (e.g. unavailability ofthe organ), chronic transplant rejection inevitably leads to the deathof the patient.

In immunological terms, the acute rejection of transplanted organs (notto be confused with the substantially less frequent, hyper-acuterejection reaction, which is antibody-mediated) represents a type-IVhypersensitivity reaction (delayed reaction type or delayed typehypersensitivity). The antigen (generally histocompatibility antigens onthe endothelial cells lining the blood vessels of the donor organ) isphagocyted by (tissue) macrophages, processed and presented to T-helpercells (CD4-positive); the sensitisation of the T-helper cells lasts forseveral days. On the second contact, the T-helper cells sensitised inthis manner are transformed into Th1 cells. In this context, theCD154-ligand-mediated co-stimulation of the antigen-presenting cell(this expresses the corresponding CD40-receptor) plays an important rolebecause interleukin-12 is released from the macrophages via this signalpathway. Interleukin 12 initiates the differentiation and proliferationof the T-helper cells. For their part, the Th1-cells stimulate theformation of monocytes in the bone marrow via given growth factors (e.g.granulocyte-macrophage colony-stimulating factor), recruit these withthe assistance of given chemokines (e.g. macrophage migration inhibitoryfactor [MIF]) and activate them via the release of interferon-γ. Theresulting very severe inflammatory reaction can destroy the transplantedtissue to great extent. CD8-positive cytotoxic T-cells, which destroytheir target cells by cytolysis and/or by inducing programmed cell deathalso participate in transplant rejection. Like the CD4-positiveTh1-cells, cytotoxic T-cells can only recognise their target (theforeign cell surface) through prior antigen presentation and by “arming”themselves accordingly. In this context, CD154-CD40-mediatedco-stimulation is also important. According to the latest knowledge, theendothelial cells of the donor organ themselves possibly have antigenpresenting and co-stimulatory properties.

Comparable with transplant rejection, but in the reverse direction isgraft-versus-host disease (GvHD), which occurs in the course ofallogenic bone-marrow transplantation (between genetically non-identicalindividuals) in approximately 40% of recipients. In the acute phaselasting up to three months, the T-cells of the donor, transferred withthe stem cells, attack the host organism; the resulting, sometimessevere inflammatory reaction manifests itself by preference in the skinand less frequently in the gastrointestinal tract and in the liver.Immunosuppression, with the potentially serious side-effects alreadydescribed, is therefore also indicated in these patients. Once again,endothelial cells, this time those of the recipient organism,participate in the initiation of this inflammatory reaction. Alongsideacute GvHD, there is also the chronic form, which requires a moreprolonged immunosuppression.

The immunosuppressants used for the prevention of acute transplantrejection generally vary in dependence upon the organ type and/or arelicensed for immunosuppression only after transplantation of certainorgans. A typical treatment for recipients of a heart transplant is thecombination of cyclosporin A with azathioprine and cortisone.Cyclosporin is increasingly being replaced by tacrolimus andazathioprine is being replaced by mycophenolate mofetil. Cyclosporin A,rapamycin and tacrolimus inhibit the T-cell activation; azathioprine andmycophenolatmofetil are antimetabolites; and corticosteroids act in ananti-inflammatory manner by inhibiting gene expression. In spite oftheir undisputed therapeutic effects, life-long systemic treatment withthese drugs is inevitably associated with sometimes seriousside-effects. In particular, these include myelotoxicity, neurotoxicity,nephrotoxicity, metabolic disorders even including the induction ofdiabetes mellitus, arterial hypertension, infections and malignancy.GvHD is generally also treated with the drugs named above, frequently incombination.

Antigen-presenting cells and T-cells communicate inter alia viaCD40-receptors and CD154-ligand, and this co-stimulation plays animportant role, in the context of transplant rejection and also GvHD, inthe Th1-cell-mediated inflammatory reaction and/or the activation ofcytotoxic T-cells. Like antigen-presenting cells, endothelial cells alsoconstitutively express the CD40 receptor. The interaction of theendothelial cells with CD154-expressing T-helper cells (naive T-helpercells and/or activated Th1-cells) results in an increased cellularexpression of chemokines and adhesion molecules. As a result, there isan increase in the recruitment and activation of circulating monocytes;these emigrate into the vascular wall and are differentiated intomacrophages. Moreover, by contrast with other antigen-presenting cells,the endothelial cells release biologically active interleukin-12exclusively after CD40 activation. Interleukin-12 is the most importantfactor for the differentiation of naive T-helper cells into Th1-cellsand promotes the subsequent clonal expansion (proliferation) of theTh1-cells. The intensified formation of interferon-γ by thedifferentiated Th1-cells stimulates not only the activity of theinfiltrated macrophages, but also intensifies the expression of CD40 inthe endothelial cells. A vicious circle can develop as a result, inwhich the endothelial cells, T-helper cells and macrophages stimulateone another, thereby maintaining the inflammatory reaction, whichdamages the transplant (acute transplant rejection) and or the recipientorganism (GvHD).

In this context, the blockade of the CD154/CD40-mediated co-stimulationpresents a promising goal for reducing and/or inhibiting acutetransplant rejection. The results from animal experiments indicate thatthe antibody-supported neutralisation of CD154 immediately followingtransplantation can even produce immunotolerance. Furthermore, chronictransplant rejection (transplant atherosclerosis) is favourablytherapeutically influenced by this intervention. One disadvantage ofthis antibody therapy is, inter alia, the danger of hypersensitivityreactions (to the antibody), above all in the case of repeatedapplication, and the poor accessibility at least for tissue-boundantigens (e.g. T-cells which have emigrated into the wall of the bloodvessel of the donor organ), because the antibodies must generally beapplied through the blood and then fail at the endothelial cell barrier.

The present invention is therefore based upon the object of providingmeans for a prevention and/or treatment of acute and chronic transplantrejection including GvHD and the associated consequences for morbidityand mortality of the affected patients. This object is achieved by thesubject matter defined in the claims.

The invention is explained in greater detail with reference to thefollowing diagrams.

FIG. 1 shows, in the form of a bar chart, the effects of an AP-1cis-element decoy (SEQ ID NO: 3) and a mutated control oligonucleotide(mut) on the basal CD40 protein expression in resting human endothelialcells, which were incubated for 8 hours with the correspondingoligonucleotide (10 μM) (n=7-10, statistical summary, related as apercentage to the basal expression, *P<0.05 versus basal; tP<0.05 versuscis-element decoy). The representative Western-blot analysis shows theeffects (4 and/or 8 hours incubation) of the nucleic acids used on thebasal CD40-protein content in resting cells. β-actin (internal standard)is used to demonstrate that identical quantities of protein wereanalysed.

FIG. 2 shows, in a representative Western-blot analysis, the effects ofselected AP-1 cis-element decoys (SEQ ID NO: 3, 5, 11, 13 and 35) and ofa mutated control oligonucleotide (mut) on the basal CD40-proteinexpression in resting human endothelial cells, which were incubated for8 hours with the corresponding cis-element decoys (10 μM). The relativeintensities (%), measured by densiometric evaluation (One-Dscan-Gelanalysis software, Scanalytics, Billerica, Mass., USA), are indicatedwith reference to the maximum value of the CD40-protein content inendothelial cells, which were not incubated with an AP-1 cis-elementdecoy.

FIG. 3 shows, in the form of a bar chart and a representativeWestern-blot analysis with β-actin as the internal standard, the effectof the AP-1 cis-element decoy SEQ ID NO: 3 by comparison with theabsence of effect of an NFAT (nuclear factor of activated T-cells)cis-element decoy on the basal CD40-protein expression in resting humanendothelial cells, which were incubated for 8 hours with thecorresponding oligonucleotide (10 μm). Statistical summary (n=4, relatedas a percentage to the basal expression; *P<0.05 versus basal; tP<0.05versus AP-1 cis-element decoy).

FIG. 4 shows, in the form of a linear graph, the effect of long-termexposure (empty circles) or respectively of a two-hour preliminaryincubation (filled circles) with the AP-1 cis-element decoy SEQ ID NO: 3(10 μM) on the basal CD40-protein expression in resting humanendothelial cells over a period of 24 hours (n=3-7).

FIG. 5 shows, in the form of a bar chart and an RT-PCR analysis, theeffects of a preliminary incubation (4 hours, 10 μM) with the AP-1cis-element decoy SEQ ID NO: 3 or respectively of a mutated controloligonucleotide (mut) on the subsequent CD40 ligand- (exposure to CD40ligand-expressing jurkat-T-cells) induced IL12-p mRNA expression inhuman endothelial cells (n=3). Representative RT-PCR analysis andstatistical summary (related as a percentage to the maximum value ofIL-12-p40 expression with CD154 stimulation, *P<0.05 versus CD154;tP<0.05 versus AP-1 cis-element decoy).

FIG. 6 shows, in the form of a bar chart, the effect of the AP-1cis-element decoy SEQ ID NO: 3 by comparison with the controloligonucleotide (mut) on the adhesion of human THP-1 monocytes to humanendothelial cells, which were pre-incubated for 8 hours with thecorresponding oligonucleotide (10 μM) and then co-cultivated for 12hours with human CD154-transfected mouse myeloma cells (P3xTB.A7, CD154)(statistical summary n=10-13, *P<0.05 versus CD154; tP<0.05 versus AP-1cis-element decoy). Non transfected P3xTB.A7 cells (−CD154) wereincluded as a negative control. Before the start of the THP-1-cellperfusion, the myeloma cells were almost completely removed from theendothelial cells in a washing stage with the medium.

FIG. 7 shows, in a representative electrophoretic mobility-shiftanalysis (EMSA), the effect of a 50-fold surplus of selected AP-1cis-element decoys (SEQ ID NO: 3, 5, 11, 13 and 35) by comparison with amutated control oligonucleotide (mut) on the formation of DNA proteincomplexes between a ³²P-marked oligonucleotide (11 fmol), which bindsspecifically to the transcription factor AP-1, and a nuclear proteinpreparation from human THP-1 monocytes in a 15 μl reaction mixture.

FIG. 8 shows, in a representative EMSA, the effect of selected AP-1cis-element decoys (SEQ ID NO: 3, 5 and 13) and of a mutated controloligonucleotide (mut) on the translocation of AP-1 into the nucleus ofhuman endothelial cells, which were incubated for 4 hours with thecorresponding cis-element decoy (10 μM). Representative EMSA, whichconfirms the cellular absorption (and action) of the various cis-elementdecoys in human endothelial cells. Comparable results were obtained inat least two other independent experiments.

FIG. 9 shows, in a representative RT-PCR, the effects of a preliminaryincubation (4 hours, 10 μM) with selected AP-1 cis-element decoys (SEQID NO: 3, 5 and 11) on the MCP-1 (monocyte chemoattractant protein-1)expression in human endothelial cells, which were incubated for 6 hourswith 60 U/ml interleukin-1β (IL-1β). Representative RT-PCR (the relativeintensities (%), measured by densiometric evaluation, are indicatedrelative to the maximum value with IL-1β stimulation).

FIG. 10 shows, in the form of a bar chart and a representativeWestern-blot analysis, the effect of an AP-1 cis-element decoy (SEQ IDNO: 3) and a mutated control oligonucleotide (mut) on the basalCD40-protein expression in isolated endothelial-intact segments from therat aorta, which were incubated in Waymouth medium. The cis-elementdecoys (10 μM) were added to the incubation medium after 1 hourpre-incubation and incubated for 11 hours with the vascular segments(6-8 segments from 5 different animals; statistical summary, related asa percentage to the basal expression, *P<0.05 versus basal; tP<0.05versus cis-element decoy). The representative Western-blot analysisshows, by way of example, that the CD40-protein in the vascular segmentsinvestigated is primarily localised in the endothelial cells and thatits expression can be significantly increased by adding the cytokinetumour necrosis factor-α (TNF-α, 1000 U/ml) and interferon-γ (IFNγ, 100U/ml) to the incubation medium for 12 hours. The detection of β-actin(internal standard) is used to demonstrate that identical quantities ofprotein were analysed.

The terms “decoy oligonucleotide” and “cis-element decoy” as used in thepresent document refer to a double-strand DNA molecule, which provides asequence, to which the transcription factor AP-1 binds in the cell, andwhich corresponds to or resembles the natural AP-1 core-binding sequencein the genome (derivative). The cis-element decoy therefore acts as amolecule for the competitive inhibition (neutralisation) of AP-1.

Transcription factors are DNA-binding proteins, which are deposited inthe cell nucleus on the promoter region of one or more genes andtherefore control their expression; that is to say, the new formation ofthe proteins, for which this gene codes. Alongside the physiologicallyimportant control of development and differentiation processes in thehuman body, transcription factors have a major pathogenic potential,primarily if they activate gene expression at the wrong time.Additionally, (under some circumstances, the same) transcription factorscan block genes with a protective function and therefore act in apredisposing manner for the formation of a disease.

The present invention therefore consists in the provision of a decoyoligonucleotide, which is capable of binding in a sequence-specificmanner to the transcription factor activator or activating protein-1(AP-1) and which has one of the following sequences. Only one strand ofthe decoy oligonucleotide is shown here, but the complementary strand isalso included: (SEQ ID NO:1) 5′-VTGAGTCAS-3′, where V = A, C or G and S= C or G (SEQ ID NO:2) 5′-STGACTCAB-3′, where S = C or G and B = G, C orT (SEQ ID NO:3) 5′-CGCTTGATGACTCAGCCGGAA-3′, (SEQ ID NO:5)5′-GTGCTGACTCAGCAC-3′, (SEQ ID NO:7) 5′-GTGGTGACTCACCAC-3′, (SEQ IDNO:9) 5′-AGTGGTGACTCACCACT-3′, (SEQ ID NO:11) 5′-TGTGCTGACTCAGCACA-3′,(SEQ ID NO:13) 5′-TTGTGCTGACTCAGCACAA-3′, (SEQ ID NO:15)5′-TGGTGAGTCACCA-3′, (SEQ ID NO:17) 5′-ATGGTGAGTCACCAT-3′, (SEQ IDNO:19) 5′-TATGGTGAGTCACCATA-3′, (SEQ ID NO:21)5′-CTATGGTGAGTCACCATAG-3′, (SEQ ID NO:23) 5′-CCTATGGTGAGTCACCATAGG-3′,(SEQ ID NO:25) 5′-TGTTGAGTCACCA-3′, (SEQ ID NO:27)5′-GTGTTGAGTCACCAC-3′, (SEQ ID NO:29) 5′-TGTGTTGAGTCACCACA-3′, (SEQ IDNO:31) 15′-CTGTGTTGAGTCACCACAG-3′, (SEQ ID NO:33)5′-ACTGTGTTGAGTCACCACAGT-3′, (SEQ ID NO:35) 5′-GTCGCTTAGTGACTAAGCGAC-3′,

The inventors surprisingly discovered that neutralisation of thetranscription factor AP-1 using corresponding decoy oligonucleotidesleads within a few hours to a decline in CD40-expression in humancultivated endothelial cells (FIGS. 1-4) and also in native ratendothelial cells (FIG. 10). This effect occurred after approximately 4hours and endured for at least 10 hours (FIGS. 1 and 4). It was alsounexpected and surprising that this effect became apparent almostsimultaneously and to largely the same extent at the mRNA and proteinlevels. However, decoy oligonucleotides, which are directed againstother transcription factors (e.g. nuclear factor of activated T-cells,NFAT), do not influence the constitutive CD40 expression (FIG. 3).Control oligonucleotides, which provide an identical sequence to theAP-1 consensus core binding sequence (SEQ ID NO: 1 and 2) apart from oneor two bases, also did not show this effect (FIGS. 1 and 2).Furthermore, a two-hour exposure of the endothelial cells to the AP-1decoy oligonucleotide was adequate to suppress the CD40 expression (FIG.4).

One consequence of the AP-1-decoy-oligonucleotide-mediated reduction ofthe CD40 protein content in the endothelial cells was a markedinhibition of the CD154-induced new formation of interleukin-12 p40(FIG. 5), the rate-determining step in the synthesis of biologicallyactive interleukin-12 (Lienenlüke et al. (2000) Eur. J. Immunol. 30,2864). The CD154-induced expression of the vascular cell adhesionmolecule-1 (VCAM-1) was also reduced to a comparable extent (58%inhibition), and in agreement with this, the CD154-induced intensified(and primarily VCAM-1 mediated) adhesion of THP-monocytes to theendothelial cells (FIG. 6).

AP-1(http://www.cbil.upenn.edu/cgi-bin/tess/tess33?request=FCT-DBRTRV-Accno&key=T00029)is among the group, comprising approximately 46 members, of theso-called basic region leucine zipper or bZIP transcription factors. Theactive transcription factor generally consists of a jun/jun-homodimer ora jun/fos-heterodimer. Both fos (Genbank Accession Number V01512) andalso jun (GenBank Accession Number J04111) must, for this purpose, bephosphorylated via corresponding protein kinases within the context ofthe cell activation, wherein the activation of this fos-kinase orrespectively jun-kinase once again depends upon the activity of otherprotein kinases disposed higher in the signal transduction pathway (e.g.protein kinase C or stress-activated protein kinase, SEK-1). Generallyin conjunction with other transcription factors, AP-1 plays an importantrole in the expression of a plurality of immuno-relevant genes such asinterleukin-2, interleukin-4, interleukin-8, interferon-γ, MCP-1, MIFand tumour necrosis factor-α. A blockade of the activity of AP-1 cantherefore interfere, for example, with the interleukin-2-dependentautocrine stimulation of T-cells and their clonal expansion.

To avoid a general weakening of the specific (cellular or respectivelyhumoral) immune defences, the decoy oligonucleotide according to theinvention is therefore preferably applied locally rather thansystemically. The ex vivo treatment of a donor organ or a bone marrowdonation before the transplantation represent preferred indications.

In this context, it is particularly important that the decoyoligonucleotides according to the invention become active immediatelyafter absorption into the target cells; by contrast, the efficacy ofantisense or RNA-interference oligonucleotides is primarily dependentupon the conversion of the protein in the cell and therefore upon itsre-synthesis.

By contrast with the use of a corresponding control decoyoligonucleotide, if a decoy oligonucleotide according to the inventionis used against AP-1 in human endothelial cells, the expression of CD40is significantly reduced by more than 50%. Moreover, switching off theAP-1 activity leads to a highly significant inhibition of theCD154-stimulated expression of interleukin-12 p40 or respectivelyVCAM-1. This leads, within the context of transplant rejection orrespectively GvHD, to a significant weakening of the endothelial T-cellinteraction or respectively endothelial-monocyte interaction, but alsoof the T-cell interaction with other antigen-presenting cells(macrophages, dendritic cells and B-lymphocytes).

These effects of a decoy oligonucleotide against AP-1 (SEQ ID NO: 3)were unambiguously confirmed in a model of acute transplant rejection(heterotopic heart transplantation in rats). However, the correspondingmutated control oligonucleotide (SEQ ID NO: 47) showed no therapeuticeffect and therefore illustrated the specificity of this therapeuticapproach. The more than 60% inhibition of chronic transplant rejectionin the same animal-experiment model by short-term exposure of thecoronary arteries of the transplant to AP-1 cis-element decoy before theimplantation was, on this scale, even more impressive and unexpected.

Accordingly, the use of the double-strand DNA oligonucleotides accordingto the invention, also referred to as decoy oligonucleotides orcis-element decoys, which contain a consensus core-binding position forAP-1, represents the preferred method for specific inhibition of AP-1activity. The exogenous supply of a large number of transcription-factorbinding positions to a cell, especially in a considerably larger numberthan present in the genome, produces a situation, in which the majorityof a given transcription factor binds specifically to the relevantcis-element decoy and not to its endogenous target-binding positions.This approach to inhibiting the binding of transcription factors totheir endogenous binding position is also referred to as squelching.Squelching (or neutralisation) of transcription factors usingcis-element decoys has been successfully used to inhibit the growth ofcells. In this context, DNA fragments were used, which containedspecific binding positions for the transcription factor E2F (Morishitaet al., PNAS, (1995) 92, 5855).

The sequence of nucleic acids, which is used to prevent the binding ofthe transcription factor AP-1, is, for example, the sequence, whichbinds naturally to the AP-1 in the cell. AP-1 binds specifically to themotif with the sequence 5′-VTGAGTCAS-3′ (SEQ ID NO:1), where V=A, C or Gand S=C or G. An effective binding of AP-1 depends upon the exactagreement with this sequence, wherein the complementary strand5′-STGACTCAB-3′ (SEQ ID NO:2), where S=C or G and B=G, C or T, can bindthe transcription factor equally efficiently. The cis-element decoy canalso be larger than the 9-mer core-binding sequence and can be extendedat the 5′ end and/or at the 3′ end. Corresponding mutations in theregion of the core-binding sequence (e.g. 5′-VTGACTCAA-3′ or5′VTTACTTAG-3′) lead to a partial or complete loss of the binding ofAP-1 to the decoy oligonucleotide (FIG. 7). Moreover, a largelypalindromic sequence of the two DNA strands favours transport into thetarget cells without auxiliary agents. Apart from the generalrequirement that the decoy oligonucleotides according to the inventioneffectively neutralise the transcription factor AP-1 in vitro, it iscritical for therapeutic efficacy that the DNA molecule is absorbedrapidly and to an adequate extent into the target cell. This isvisualised by the differential effect of neutralisation of AP-1 by thesame decoy oligonucleotides in a cell-free system (FIG. 7) by comparisonwith intact cells (FIG. 8) or respectively their effect on geneexpression in these cells (inhibition of the IL-1β stimulated expressionof MCP-1; FIG. 9). Moreover, the cis-element decoy according to theinvention should not exceed a given length, because this has a limitingeffect on the transport into the target cell. Every decoyoligonucleotide with a length of at least 9 bp (consensus core bindingsequence) up to a length of approximately 45 bp is suitable, preferablyup to a length of 27 base pairs, by particular preference up to a lengthof approximately 23 base pairs, by particular preference with a lengthof 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 basepairs.

Since the cis-element decoy is a double-strand nucleic acid, each DNAoligonucleotide according to the invention comprises not only the senseor forward sequence but also the complementary antisense or reversesequence. Preferred DNA oligonucleotides according to the invention havea 9-mer core-binding sequence for AP-1, as contained in SEQ ID NO: 1.However, the cis-element decoy can also have a sequence different fromthe above sequence and can be longer than a 9-mer. Sequences ascontained in SEQ ID NO: 3 to SEQ ID NO: 36 are particularly preferred.This listing of the preferred sequences is not finite. It is evident toa person skilled in the art that a plurality of sequences can be used asinhibitors for AP-1, so long as they fulfil the conditions of the 9-merconsensus core-binding sequence listed above and have an affinity forAP-1.

The affinity of the binding of a nucleic acid sequence to AP-1 can bedetermined by Electrophoretic Mobility Shift Assay (EMSA) (Sambrook etal. (1989) Molecular Cloning. Cold Spring Harbor Laboratory Press;Krzesz et al. (1999) FEBS Lett. 453, 191). This test system is suitablefor quality control of nucleic acids which are intended for use in themethod according to the present invention, or for determining theoptimum length of a binding position. It is also suitable for theidentification of other sequences which are bound by AP-1.

The method of the present invention modulates the transcription of agene or genes in such a manner that the gene or genes, e.g. CD40, areexpressed to a reduced extent or not at all. Reduced or suppressedexpression within the context of the present invention means that thetranscription rate is reduced by comparison which cells which have notbeen treated with a decoy oligonucleotide according to the invention. Areduction of this kind can be detected, for example, by Northern-blot(Sambrook et al., 1989) or RT-PCR (Sambrook et al., 1989). A reductionof this kind is typically a 2-fold, especially at least a 5-fold, inparticular, at least a 10-fold reduction.

The loss of activation can be achieved, for example, if AP-1 acts with agiven gene as a transcription activator, and accordingly, squelching ofthe activator leads to the loss of expression of the target gene.However, an indirect inhibition is also possible by modulating themRNA-(post-transcriptional effect) or protein instability(post-translational effect) of the target gene. In this case, theneutralisation of AP-1 would lead, for example, to a reduced expressionof proteins which prevent the breakdown of the mRNA of the target geneby RNases and/or which prevent the proteolytic degradation of the targetprotein. An effect of this kind seems to participate in the effectdescribed above of the AP-1 cis-element decoys on the CD40-expression inthe human endothelial cells. Oligonucleotides are generally rapidlybroken down by endonucleases and exonucleases, in particular, DNases andRNases in the cell. Accordingly, the DNA oligonucleotides can bemodified in order to stabilise them against degradation, so that, a highconcentration of the oligonucleotides is maintained within the cell overa relatively long period. Typically, a stabilisation of this kind can beobtained by the introduction of one or more modified internucleotidebonds.

A successfully stabilised DNA oligonucleotide does not necessarilycontain a modification at every internucleotide bond. Theinternucleotide bonds at each end of both oligonucleotides of thecis-element decoy are preferably modified. In this context, the lastsix, five, four, three, two, the last one, or one or moreinternucleotide bonds within the last six internucleotide bonds can bemodified. Furthermore, various modifications of the internucleotidebonds can be introduced into the nucleic acids, and the resulting decoyoligonucleotides can be tested for sequence-specific bonding to AP-1using the routine EMSA test system. This test system allows thedetermination of a binding constant of the cis-element decoy andtherefore allows a determination of whether the affinity has beenchanged by the modification. Modified cis-element decoys, which stillshow an adequate binding, can be selected, an adequate binding being atleast approximately 50% or at least approximately 75%, and by particularpreference approximately 100% of the binding of the un-modified nucleicacid.

Cis-element decoys with modified internucleotide bonds, which still showadequate binding, can be checked to determine whether they are morestable in the cell than un-modified cis-element decoys. The cells“transfected” with the cis-element decoys according to the invention areinvestigated at different times for the quantity of cis-element decoystill present. In this context, a cis-element decoy marked with afluorescence-dye (e.g. Texas-Red) or a radioactively marked (e.g. ³²P or³⁵S) cis-element decoy is preferably used with subsequentdigital-fluorescence microscopy and/or autoradiography or scintigraphy.A successfully modified cis-element decoy has a half-life in the cell,which is greater than that of an un-modified cis-element decoy,preferably of at least approximately 48 hours, by greater preference atleast approximately 4 days, by greatest preference at leastapproximately 7 days.

Suitable modified internucleotide bonds are summarised in Uhlmann andPeyman ((1990) Chem. Rev. 90, 544). Modified internucleotide phosphateresidues and/or a non-phosphorus bridges in a nucleic acid, which can beused in a method according to the present invention, contain, forexample, methylphosphonate, phosphorothioate, phosphorodithioate,phosphoroamidate, phosphate ester; while non-phosphorus internucleotideanalogues contain, for example, siloxane bridges, carbonate bridges,carboxymethylester bridges, acetamidate bridges and/or thioetherbridges. When using phosphorothioate-modified internucleotide bonds,these should preferably not be disposed between the bases cytosine andguanine, because this can lead to an activation of the target cells ofthe cis-element decoy.

One further embodiment of the invention is the stabilisation of nucleicacids through the introduction of structural features into the nucleicacids, which increase the half-life of the nucleic acid. Structures ofthis kind, which contain hairpin and dumbbell DNA, are disclosed in U.S.Pat. No. 5,683,985. At the same time, modified internucleotide phosphateresidues and/or non-phosphorus bridges can be introduced together withthe structures named. The resulting nucleic acids can be tested forbinding and stability in the test system described above.

A cis-element decoy of the present invention is absorbed rapidly intothe cell. An adequate absorption is characterised by the modulation ofthe expression of one or more genes, which are subject to control byAP-1 (e.g. CD40). The cis-element decoy of the present inventionpreferably modulates the transcription of a gene or genes afterapproximately 4 hours of contact with the cell, by greater preferenceafter approximately 2 hours, after approximately 1 hour, afterapproximately 30 minutes and by the greatest preference afterapproximately 10 minutes. A typical mixture which is used in anexperiment of this kind, contains 10 μmol/l cis-element decoy.

Furthermore, the present invention relates to the use of the decoyoligonucleotides according to the invention for the manufacture of apharmaceutical agent, especially for the prevention and/or treatment ofacute and chronic transplant rejection, acute and chronicgraft-versus-host disease (GvHD) and ischaemic/reperfusion damage toorgans following a surgical intervention.

Moreover, the invention relates to a method for modulating thetranscription of at least one gene in cells, especially in endothelialcells and antigen-presenting cells (monocytes, macrophages, dendriticcells, B-cells), wherein the method comprises the step of bringing thenamed cells into contact with a mixture containing one or moredouble-strand nucleic acid(s) according to the invention, which arecapable of binding in a sequence-specific manner to the transcriptionfactor AP-1. One preferred method is, for example, the ex vivo treatmentof a donor organ before introducing it into the body of the recipient byapplying the nucleic-acid-containing mixture into the blood vessels ofthe donor organ (orthograde or retrograde).

The mixture containing the cis-element decoys according to the inventionis brought into contact with the target cells (e.g. endothelial cells,epithelial cells, leukocytes, smooth muscle cells, keratinocytes orfibroblasts). The purpose of this contacting is to transfer thecis-element decoys, which bind AP-1, into the target cell (for example,the AP-1-dependent CD40-expressing cell). Accordingly, nucleic acidmodification and/or additives or auxiliary substances, of which it isknown that they increase the penetration of membranes, can be usedwithin the framework of the present invention (Uhlmann and Peyman (1990)Chem. Rev. 90, 544).

In one preferred embodiment, a mixture according to the inventioncontains essentially only nucleic acid and buffer. An appropriateconcentration of the cis-element decoy is within the range of at least0.1 to 100 μM, preferably approximately 10 μM, wherein one or moresuitable buffers can be added. An example of a suitable buffer is amodified Ringer's solution containing 145 mmol/l Na⁺, 5 mmol/l K⁺, 50mmol/l Cl⁻, 2 mmol/l Ca²⁺, 1 mmol/l Mg²⁺, 10 mmol/l Hepes, 106 mmol/lisethionate, 10 mmol/l D-glucose, pH 7.4.

In a further embodiment of the invention, the mixture additionallycontains at least one additive and/or auxiliary agent. Additives and/orauxiliary agents such as lipids, cationic lipids, polymers, liposomes,nanoparticles, nucleic acid-aptamers, peptides and proteins, which arebound to DNA, or synthetic peptide-DNA molecules are intended, forexample, to increase the introduction of nucleic acids into the cell, todirect the mixture towards only one sub-group of cells, to prevent thebreakdown of the nucleic acid in the cell, to facilitate the storage ofthe nucleic acid mixture before use. Examples of peptides and proteinsor synthetic peptide-DNA molecules are, for example, antibodies,antibody fragments, ligands, adhesion molecules, all of which can bemodified or un-modified.

Additives, which stabilise the cis-element decoys in the cell are, forexample, nucleic-acid-condensing substances, such as cationic polymers,poly-L-lysine or polyethylenimine.

The mixture, which is used in the method of the present invention, ispreferably applied locally by injection, infusion, catheter, pluronicgels, polymers, which provide a prolonged release of medicines, or anyother device, which allows local access. The ex vivo application of themixture (infusion and/or incubation) used in the method of the presentinvention, also allows local access.

The following drawings and examples are provided only by way ofexplanation and in no sense restrict the scope of the invention.

1. Cell Culture

Human endothelial cells were isolated from umbilical veins by treatmentwith 1.6 U/ml dispase in Hepes-modified tyrode solution for 30 minutesat 37° C. and cultivated on gelatine-coated 6-well tissue-culture dishes(2 mg/ml gelatine in 0.1 M HCl for 30 minutes at room temperature) in1.5 ml M199 medium (Gibco Life Technologies, Karlsruhe, Germany),containing 20% foetal calf serum, 50 U/ml penicillin, 50 μg/mlstreptomycin, 10 U/ml nystatin, 5 mM HEPES and 5 mM TES, 1 μg/ml heparinand 40 μg/ml endothelial growth factor. They were identified by theirtypical pavement morphology, positive immuno-staining for vonWillebrandt factor (vWF) and fluorimetric detection (FACS) of PECAM-1(CD31) and negative immuno-staining for smooth muscular α-actin (Krzeszet al. (1999) FEBS Lett. 453, 191).

The human monocyte cell line THP-1 (ATCC TIB 202), the human jurkat cellline D1.1 (ATCC CRL-10915) and the mouse myeloma cell line P3xTB.A7 werecultivated in RPMI 1640 medium (Life technologies), containing 10%foetal calf serum, 50 U/ml penicillin, 50 μg/ml streptomycin and 10 U/mlnystatin.

2. RT-PCR Analysis

The total endothelial RNA was isolated using the Qiagen RNeasy Kit(Qiagen, Hilden, Germany), and following this, a cDNA synthesis wascarried out with a maximum of 3 μg RNA and 200 U Superscript™ II reversetranscriptase (Life Technologies) in a total volume of 20 μl inaccordance with the manufacturer's instructions. For the calibration ofthe cDNA charge, 5 μl (approximately 75 ng cDNA) of the resulting cDNAsolution and the primer pair (Gibco) for the elongation factor-1(EF-1)-PCR with 1 U Taq DNA polymerase (Gibco) were used in a totalvolume of 50 μl. EF-1 was used as an internal standard for the PCR. ThePCR products were separated on 1.5% agarose gels containing 0.1%ethidium bromide and the intensity of the bands was measureddensiometrically with a CCD camera system and the One-Dscan gel analysissoftware by Scanalytics (Billerica, Mass., USA), in order to adapt thevolume of the cDNA in subsequent PCR analyses.

All PCR reactions were carried out individually for each primer pair ina Hybaid OmnE Thermocycler (AWG, Heidelberg, Germany). The individualPCR conditions for the cDNA of human endothelial cells were as follows:CD40 (product size 381 bp, 25 cycles, addition temperature 60° C.,(forward primer) 5′-CAGAGTTCACTGAAACGGAATGCC-3′ (SEQ ID NO: 37),(reverse primer) 5′-TGCCTGCCTGTTGCACAACC-3′ (SEQ IS NO: 38); EF-1(product size 220 bp, 22 cycles, addition temperature 55° C., (forwardprimer) 5′-TCTTAATCAGTGGTGGAAG-3′ (SEQ ID NO: 39), (reverse primer)5′-TTTGGTCAAGTTGTTTCC-3′ (SEQ ID NO: 40); IL-12p40 (product size 281 bp,30 cycles, addition temperature 62° C., (forward primer)5′-GTACTCCACATTCCTACTTCTC-3′ (SEQ ID NO: 41), (reverse primer)5′-TTTGGGTCTATTCCGTTGTGTC-3′ (SEQ ID NO: 42); MCP-1 (product size 330bp, 22 cycles, addition temperature 63° C., (forward primer)5′-GCGGATCCCCTCCAGCATGAAAGTCTCT-3′ (SEQ ID NO: 43), (reverse primer)5′-ACGAATTCTTCTTGGGTTGTGGAGTGAG-3′ (SEQ ID NO: 44); VCAM-1 (product size523 bp, 26 cycles, addition temperature 63° C.), (forward primer)5′-CATGACCTGTTCCAGCGAGG-3′ (SEQ ID NO: 45), (reverse primer)5′-CATTCACGAGGCCACCACTC-3′ (SEQ ID NO: 46).

3. Electrophoretic Mobility Shift Assay (EMSA)

The nuclear extracts and [³²p]-marked double-strand consensusoligonucleotides (Santa Cruz Biotechnologie, Heidelberg, Germany),non-denatured polyacrylamide gel electrophoresis, autoradiography andsupershift analysis were implemented as described in Krzesz et al.(1999) FEBS Lett. 453, 191. In this context, a double-strand DNAoligonucleotide was used with the following single strand sequence (corebinding sequence is underlined): AP-1, 5′-CGCTTGATGACTCAGCCGGAA-3′ (SEQID NO: 3). For the analysis of the displacement of endogenous AP-1 innuclear extracts of the endothelial cells by the various cis-elementdecoys, a ratio of 50:1 AP-1 cis-element decoy: [³²p]-marked AP-1oligonucleotide (11 fmol)) was selected in the EMSA binding mixture.

4. Decoy Oligonucleotide Technique

Double-strand decoy oligonucleotides were manufactured from thecomplementary single-strand phosphorothioate-linked oligonucleotides(Eurogentec, Köln, Germany) as described in Krzesz et al. (1999) FEBSLett. 453, 191. The cultivated human endothelial cells were incubatedfor at least 2 hours with the relevant decoy oligonucleotide in aconcentration of 10 μM. Following this, thedecoy-oligonucleotide-containing medium was generally replaced withfresh medium. The single-strand sequences of the oligonucleotides wereas follows (underlined letters indicate phosphorothioate-linked bases):(SEQ ID NO:3) AP-1 5′-CGCTTGATGACTCAGCCGGAA-3′ (SEQ ID NO:5) AP-15′-GTGCTGACTCAGCAC-3′ (SEQ ID NO:11) AP-1 5′-TGTGCTGACTCAGCACA-3′ (SEQID NO:13) AP-1 5′-TTGTGCTGACTCAGCACAA-3′ (SEQ ID NO:35) AP-15′-GTCGCTTAGTGACTAAGCGAC-3′ (SEQ ID NO:47) AP-1 mut5′-CGCTTGATTACTTAGCCGGAA-3′ (SEQ ID NO:48) NFAT5′-CGCCCAAAGAGGAAAATTTGTTTCATA-3′

5. Western-Blot Analysis

The human umbilical vein endothelial cells were opened by freezingsuccessively five times in liquid nitrogen and thawing at 37° C. Proteinextracts were manufactured as described by Hecker et al. (1994) BiochemJ. 299, 247. 20-30 μg protein were separated according to a standardprotocol using a 10% polyacrylamide gel electrophoresis under denaturingconditions in the presence of SDS and transferred to a BioTrace™polyvinylidene fluoride transfer membrane (Pall Corporation, Rossdorf,Germany). A polyclonal primary anti-human-CD 40 antibody by ResearchDiagnostics Inc., Flanders N.J., USA was used for the immunologicaldemonstration of the CD40 protein. The protein bands were demonstratedafter adding a peroxidase-linked anti-rabbit IgG (1:3000, Sigma,Deisenhofen, Germany) using the chemiluminescence method (SuperSignalChemiluminescence Substrate; Pierce Chemical, Rockford, Ill., USA) andsubsequent autoradiography (Hyperfilm™ MP, Amersham Pharmacia Biotech,Buckinghamshire, England). The application and transfer of identicalprotein quantities was demonstrated after “stripping” the transfermembrane (5 minutes 0.2 N NaOH, followed by 3×10 minutes washing withH₂O) by demonstrating identical protein bands of β-actin with amonoclonal primary antibody and a peroxidase-linked anti-mouse IgG (bothfrom Sigma-Aldrich, 1:3000 dilution).

6. Endothelial Cell—Leukocyte Interaction

Primary cultivated human endothelial cells, grown on cover slips up to acell density of 100%, were washed with Hepes-tyrode buffer (data inmmol/l: NaCl 137, KCl 2.7, CaCl₂ 1.4, MgCl₂ 0.25, NaH₂PO₄ 0.4, Na-Hepes10, D-glucose 5), which contained 1.5% polyvinylpyrrolidone (PVP;Sigma-Aldrich), and applied to the base of a perfusion chamber (2.5 mmheight and 260 μl volume, Warner Instrument, Hamden, Conn., USA). Thechamber was attached to an Axiovert S100 TV microscope (Zeiss,Goettingen, Germany) on a heated platform (Warner Instruments) andperfused with the heated buffer (in-line solution heater, WarnerInstruments) at 37° C. The shear stress produced with a pump (Ismatec,Zurich, Switzerland) was 5 dyn/cm with a shear rate of 10 s⁻¹. Theendothelial cells were initially perfused for 10 minutes withHepes-tyrode/PVP, followed by a 10-minute superfusion with 1.5×10⁶ THP-1cells (×10⁵ THP-1 cells/ml) in Hepes-tyrode/PVP. Following this, theperfusion chamber was rinsed with Hepes-tyrode/PVP. The documentation ofthe cell-cell interactions was evaluated at 20× magnification with aSPOT RT Colour-CCD camera (Diagnostic Instruments, Burroughs St.Sterling Heights, Mich., USA). Three images from different fields ofview were evaluated for each test mixture using the program MetaMorphV3.0 (Universal Imaging, West Chester, Pa., USA).

7. Statistical Analysis

Unless otherwise indicated, all data in the diagrams are shown as a meanvalue ±SEM of n experiments. The statistical evaluation was implementedby one-sided variance analysis (ANOVA) followed by a Dunnett Post Test.A P-value of <0.05 was taken as a statistically significant difference.

8. Animal Experimental Demonstration of the Decoy-Oligonucleotide Action

To demonstrate the efficacy of the decoy-oligonucleotide-based therapydeveloped in the present patent application, an animal experimentalProof-of-Concept study for the indication acute transplant rejection wascarried out with rats (strain combination Wistar Furth onto Lewis;experimental details, see Hölschermann et al. (1999) A. J. Pathol. 154,211). Single application of 10 μmol/l of the AP-1-decoy oligonucleotide(SEQ ID NO: 3), but not of the mutated control oligonucleotide (AP-1 mut(SEQ ID NO: 47), no difference by comparison with the control animals),in the coronary blood vessels of the heterotopic heart transplant (30minutes incubation prior to implantation) prolonged its survival withoutthe administration of an immunosuppressant from 6.2±0.2 to 7.6±0.4 days(n=5, P<0.05). This effect was associated with a significant weakeningof the adhesion-molecule expression (e.g. VCAM-1) in the endothelium ofthe coronary blood vessels of the donor hearts and the infiltration ofmonocytes and T-cells on post operative days 1 and 3.

The single application of the AP-1 decoy oligonucleotide also provedextremely effective in the model of transplant vasculopathy (chronicrejection). By way of deviation from the previously described, acuterejection model, the recipient animals were treated intraperitoneallywith the immunsuppressant cyclosporin A (5 mg per kg body weight perday) and the donor hearts were explanted after 100 days. The degree ofvasculopathy in the coronary arteries was determined morphometrically inaccordance with the Adams criteria and showed the following picture:isotype control (n=7), score 0.97±0.11; cyclosporin A-control (n=6),score 2.08±0.16 (P>0.001 versus isotype control); AP-1 decoyoligonucleotide (n=6), score 1.39±0.16 (P<0.0l versus cyclosporinA-control).

1. A double-stranded DNA oligonucleotide, wherein one of the two DNAstrands provides a sequence according to SEQ ID NOS: 1, 2, 7 to 10 or 15to 34, and the complementary DNA strand provides the sequencecomplementary to the these.
 2. The double-stranded DNA according toclaim 1 dispersed in a pharmaceutical medium.
 3. A method for theprevention and/or treatment of acute and chronic transplant rejection,acute and chronic graft-versus-host disease (GvHD) andischaemia/reperfusion damage of organs following a surgical interventionin a subject comprising administering to said subject a double-strandedDNA oligonucleotide according to SEQ ID NO: 1 to 36.